The intrinsic structure of glucose transporter isoforms Glut1 and Glut3 regulates their differential distribution to detergent-resistant membrane domains in nonpolarized mammalian cells Tomoko Sakyo1,2, Hiroaki Naraba1, Hirobumi Teraoka2 and Takayuki Kitagawa1,3 Pharmaceutical Research Center, Iwate Medical University, Morioka, Japan Department of Pathological Biochemistry, Medical Research Institute, Tokyo Medical and Dental University, Tokyo, Japan Department of Biochemistry and Cell Biology, National Institute of Infectious Diseases, Tokyo, Japan Keywords detergent-resistant membrane; glucose transporter 1; glucose transporter 3; mammalian glucose transporter; sorting signal Correspondence T Kitagawa, Department of Cell Biology and Molecular Pathology, Iwate Medical University, School of Pharmacy, Iwate 028-3694, Japan Fax: +81 19 698 1844 Tel: +81 19 651 5111 (Ext 5150) E-mail: tkitaga@iwate-med.ac.jp (Received 23 January 2007, revised March 2007, accepted 30 March 2007) doi:10.1111/j.1742-4658.2007.05814.x The hexose transporter family, which mediates facilitated uptake in mammalian cells, consists of more than 10 members containing 12 membrane-spanning segments with a single N-glycosylation site We previously demonstrated that glucose transporter is organized into a raft-like detergent-resistant membrane domain but that glucose transporter distributes to fluid membrane domains in nonpolarized mammalian cells In this study, we further examined the structural basis responsible for the distribution by using a series of chimeric constructs Glucose transporter and glucose transporter with a FLAG-tagged N-terminus were expressed in detergentresistant membranes and non-detergent-resistant membranes of CHO-K1 cells, respectively Replacement of either the C-terminal or N-terminal cytosolic portion of FLAG-tagged glucose transporter and glucose transporter did not affect the membrane distribution However, a critical sorting signal may exist within the N-terminal half of the isoforms without affecting transport activity and its inhibition by cytochalasin B Further shortening of these regions altered the critical distribution, suggesting that a large proportion or several parts of the intrinsic structure, including the N-terminus of each isoform, are involved in the regulation The hexose transporter family, which mediates facilitated uptake in mammalian cells, consists of more than 10 members containing 12 membrane-spanning segments with a single N-glycosylation site [1,2] (Fig 1A) Among this family, glucose transporter (Glut) is widely expressed in a variety of cells and mediates much of the basal, noninsulin-independent transport of d-glucose with high affinity Glut1’s function is thought to be mainly regulated by expression through a variety of stimuli and agents, including serum, growth factors, tumor viruses, and inhibitors of oxidative phosphorylation [3–7] This N-linked glycoprotein is trafficked post-translationally to the cell surface [8] We have investigated tumor-associated alterations in Glut expression using human cell hybrids derived from cervical carcinoma HeLa cells and normal fibroblasts [9–11], whose tumorigenicity is controlled by a putative tumor suppressor gene on chromosome 11 [12] In these studies, we found that the tumor-suppressed hybrid cells express Glut1 alone, whereas tumorigenic cell hybrids express both Glut1 and Glut3 as larger forms, probably due to modifications of N-glycosylation Abbreviations CB, cytochalasin B; 2DG, 2-deoxy-D-glucose; DRM, detergent-resistant membrane; EGFP, enhanced green fluorescent protein; ECL, enhanced chemiluminescence; FG1, FLAG-tagged Glut1; FG3, FLAG-tagged Glut3; GFP, green fluorescent protein; Glut, glucose transporter; [3H]2DG, [3H]-2-deoxy-D-glucose FEBS Journal 274 (2007) 2843–2853 ª 2007 The Authors Journal compilation ª 2007 FEBS 2843 Regulation of DRM sorting of Glut1 T Sakyo et al [9–11] However, differences in the membrane distribution and roles of these isoforms remain largely unknown Glut1 and Glut3 share many similarities in structure and function About 65% of their amino acid sequences are identical, but their C-terminal domains and the extracellular loops are distinctive [1,13] (Fig 1B) Glut3 is expressed at the cell surface of various types of cell, including neuronal cells [13] and many tumor cells [14,15] These isoforms have a high affinity for d-glucose when expressed at the cell surface [16,17] A striking difference between them is seen in cellular localization In polarized epithelial cells, such as Caco-2 and MDCK cells, Glut1 is expressed on the basolateral surface, whereas Glut3 is sorted to the apical surface [18–20] It has been well established that polarized cell membranes have structural characteristics that are regulated by a unique sorting machinery to A B Fig The topology of Glut1 and alignment of the deduced amino acid sequences of Glut1 and Glut3 (A) The predicted structure of Glut1 [1] The position of the single endogenous site of N-linked glycosylation at Asn is shown CHO indicates N-linked oligosaccharide An alignment of the deduced amino acid sequences of rabbit Glut1 [42] and human Glut3 [17] is shown in (B) The amino acids are numbered to the left and are written in the single-letter code Identical amino acid sequences are indicated by asterisks The locations of predicted transmembrane domains are indicated by gray boxes 2844 FEBS Journal 274 (2007) 2843–2853 ª 2007 The Authors Journal compilation ª 2007 FEBS T Sakyo et al play tissue-specific functions [21,22] In platelets and neuronal cells, Glut3 is also present in intracellular vesicles [3,23] These results imply distinctive roles for Glut1 and Glut3 in mammalian cells, although they remain unclear A recent study by Inukai et al found that a functional signal sorting Glut3 to the apical surface in MDCK cells lies in the C-terminal cytosolic tail [24] We have previously determined the differential membrane distribution of these Gluts in nonpolarized cells such as HeLa cells and CHO-K1 cells, and found that Glut1 distributes to a raft-like detergent-resistant membrane (DRM), whereas Glut3 localizes to a fluid membrane domain [25] DRMs are recognized as specific microdomains in the plasma membrane that are enriched with cholesterol and sphingolipids to organize an ordered lipid phase, including some proteins such as caveolin, glycosylphosphatidylinositol-anchored proteins, and tyrosine kinases Mainly due to their ordered lipid nature, these membrane domains are relatively resistant to solubilization by nonionic detergents The distribution of Glut1 within DRMs has been reported in other cell types [26,27], suggesting that it is mainly due to the intrinsic properties of Gluts The molecular mechanisms by which Gluts are differentially recruited to membrane domains of nonpolarized cells remain to be clarified Therefore, we attempted to characterize the structural determinants of Glut1 and Glut3 required for the distribution We have expressed a series of chimeric transporters utilizing various portions of Glut1 and Glut3 in CHO-K1 cells and assessed their DRM distribution Our data show that, despite apical sorting of Glut3, neither the C-terminal nor the N-terminal cytosolic tail of Glut1 contains a sorting signal This signal may exist within the N-terminal half of the membrane-spanning segments of Glut1 Results Expression and DRM distribution of FLAG-tagged Glut1 (FG1) and FLAG-tagged Glut3 (FG3) Previously, we demonstrated that Glut1 is distributed to raft-like DRMs and Glut3 is distributed to fluid membranes in a nonpolarized mammalian cell line [25] The differential distribution of these isoforms seems to be independent of cell type or the amount of protein expressed To clarify the molecular basis for the control of the differential distribution of Glut1 and Glut3, we adopted a chimeric strategy whereby different portions of Glut1 and Glut3 were spliced together and Regulation of DRM sorting of Glut1 expressed in CHO-K1 cells, which express Glut1 endogenously but not Glut3 As a means of discriminating recombinant proteins from native Glut1, cDNA constructs for either Glut1, Glut3 or the Glut1 ⁄ Glut3 chimera were prepared containing a FLAG epitope tag or a green fluorescent protein (GFP)-encoded tag at their N-termini, as described in Experimental procedures These cDNA constructs were then ligated into the expression vector pCMV and used for transient expression by lipofection in CHO cells Initially, we examined whether FG1 and FG3 are able to target DRMs and non-DRMs, respectively, in CHO cells To compare the solubility of the newly synthesized proteins in nonionic detergents, the cells, which transiently expressed FG1 and FG3 proteins, were treated with 0.5% Triton X-100 at °C, following fractionation of the solubilized (S) and insoluble (I) fractions As described previously [25], immunoblotting of these samples indicated that caveolin-1, which is a well-known marker as a detergent-insoluble component [28], was present in the 0.5% Triton X-100-insoluble fraction (Fig 2A) In contrast, tubulin-a, which is another marker for solubilization, was fully solubilized under this condition Next, we examined the distribution of recombinant FG1 and FG3 proteins, using a monoclonal antibody that recognizes a FLAG epitope (MDYKDDDDK) As expected, FG3 was fully solubilized by 0.5% Triton X-100 at °C In contrast, FG1 remained in the insoluble fraction These distributions were similar to those observed with native Glut1 as well as overexpressed Glut1 and Glut3 in CHO cells, which were detected with their respective antibodies to C-terminal peptide (data not shown) b-Actin, a component of the cytoskeleton, distributed to both DRM and non-DRM fractions of CHO cells under these conditions (Fig 2A) To directly determine the location of expressed transporter proteins on the living cell surface, recombinant GFP-tagged Glut1 and GFP-tagged Glut3 were also expressed in CHO cells These GFP-tagged proteins were detected over the entire cell surface (Fig 2B), whereas GFP proteins were found in the cytoplasm The results indicate that the tagging of the N-terminus with GFP did not impair the membrane trafficking of Gluts We also observed that GFP-tagged Glut1 was still distributed to rafts in CHO-K1 cells (data not shown) Glucose uptake by CHO cells transfected with FG1 and FG3 To examine the influence of N-terminal FLAG tagging on glucose transport, the activity of CHO cells FEBS Journal 274 (2007) 2843–2853 ª 2007 The Authors Journal compilation ª 2007 FEBS 2845 Regulation of DRM sorting of Glut1 T Sakyo et al was determined Insertion of the FLAG epitope into Glut1 or Glut3 at the N-terminus did not result in significant alterations to the transport activity, because CHO cells overexpressing these proteins exhibited an increase in glucose transport activity (Fig 2C) Glucose transport in CHO cells expressing FG1 was increased 4–6-fold as compared to that in cells transfected with or without empty vector, whereas cells transfected with FG3 showed only a 3–4-fold increase This difference in the increase in transport activity might be partly due to the level of protein expression that was detected by western blot analysis (Fig 2C) The uptake of 2-deoxyglucose (2DG) by CHO cells transfected with FG1, FG3 or control vector was inhibited by about 90% by 10 lm cytochalasin B (CB) (Fig 2D), supporting a functional carrier-mediated process through these FLAGtagged proteins Neither the N-terminus nor the C-terminus of Glut1 is needed for the DRM distribution To determine the functional domains responsible for the DRM distribution of Glut1, we generated a series of Glut1 ⁄ Glut3 chimeric mutants by replacing the corresponding domains of Glut1 with the equivalent regions of Glut3 As shown in Fig 1B, the amino acid regions that are most divergent between Glut1 and Glut3 are the N-terminus and the C-terminus, and the large intracellular loop between transmembrane domains and As a unique sorting signal for the recruitment of Glut3 to the apical membrane of polarized cells exists in the C-terminus [24], we first generated a set of FLAG-tagged Glut1 ⁄ Glut3 chimeras in which the cytosolic N-terminus and C-terminus of Glut1 were replaced by the corresponding amino acids of Glut3 (Fig 3A) These N31 and C13 chimeric proteins were transiently expressed in CHO cells, and examined in terms of their detergent solubility Both proteins were similarly retained in the insoluble fraction when the cells were treated with 0.5% Triton X-100 at °C, whereas the overproduced FG3 was fully solubilized under these conditions (Fig 3C) To examine the relevance of N31 and C13 expression to glucose transport, the activity of CHO cells was determined As shown in Fig 3B, the glucose transport activity in the cells into which N31 was transfected increased, as was the case in the FG1-transfected CHO cells The amount of 2DG taken up by C13transfected cells also increased, although it was lower than that in N31-transfected cells In any case, about 90% of this activity were inhibited by CB Thus, substitution of the N-terminus (N31) or C-terminus (C13) of Glut1 with that of Glut3 resulted in little or any change in distribution to the DRM and transport activity We also tested whether the substitution of both the N-terminus and C-terminus of Glut1 with those of Glut3, i.e N31C3 (Fig 4A), had an effect The N31C3 protein expressed in CHO cells was as insoluble as FG1, whereas FG3 was fully solubilized (Fig 4B) In any case, the distribution of endogenous Glut1 was unaffected The N-terminal membrane-spanning regions of Glut1 are needed for the DRM distribution To further define the domains responsible for the DRM distribution, additional FLAG-tagged chimeric constructs, A and B, were generated (Fig 5A) In A, amino acids 2–12 and 272–492 of Glut1, which include the N-terminal cytosolic tail and C-terminal six membrane-spanning regions but exclude a large intracellular loop, were replaced by the corresponding amino acids of Glut3 Instead, in FLAG-tagged chimera B, the C-terminal six membrane-spanning region amino acids 272–451 correspond to Glut1, and the remainder correspond to Glut3 The results are shown in Fig Fig Effect of FG1 and FG3 in CHO cells (A) Schematic composition of FG1 and FG3 Glut1 and Glut3 are in dark gray and pale gray, respectively The location of the FLAG or GFP tag is indicated by hatched areas at the N-terminus FG1 and FG3 were transfected with lipofectamine, and their expression in CHO-K1 cells was determined as described in Experimental procedures (B) CHO-K1 cells were also transfected with GFP-tagged Glut1 and Glut3, and a control GFP vector After 24 h, transfected cells were plated onto glass-based dishes and subjected to confocal fluorescence microscopy The left panels show the GFP fluorescent images, and the right panels show the differential interference images (upper, FG1; middle, FG3; lower, GFP) (C) Two days after transfection, cells were solubilized with 0.5% Triton X-100 at °C Total cell lysate (T) was separated into soluble (S) and insoluble (I) fractions by centrifugation at 13 800 g for 30 min, and each sample (10 lg of protein per lane) was subjected to SDS ⁄ PAGE and immunoblotting for FLAG, Glut1, Glut3, caveolin-1, and a-tubulin The corresponding molecular masses are indicated in kDa (D) After 48 h of transfection, the uptake of [3H]2DG was measured in the presence and absence of CB The upper panel shows relative uptake values (% of control cells), and the lower panel indicates absolute uptake values normalized to the quantity of protein expressed in CHO cells (nmoles per mg of protein per 10 min) Bars and brackets reflect the means ± SD of four determinations DMSO, dimethylsulfoxide 2846 FEBS Journal 274 (2007) 2843–2853 ª 2007 The Authors Journal compilation ª 2007 FEBS T Sakyo et al Regulation of DRM sorting of Glut1 As these chimeric constructs contain both a FLAG epitope at the N-terminus and a Glut3 epitope at the C-terminus, the efficiency with which each of these plasmids was expressed was comparable Whereas the total amounts detected with antibodies for FLAG and Glut3 were similar, demonstrating a similar efficiency B A FGl or GFP-G1 Intracellular loop GFP-G1 GFP or FLAG N C FG3 or GFP-G3 GFP-G3 GFP or FLAG C N GFP C D CHO Cell Transfection Fraction Vector T S I FG1 FG3 T S I T S I FLAG-M2 Glut3 Glut1 β-Actin Caveolin-1 α-Tubulin FEBS Journal 274 (2007) 2843–2853 ª 2007 The Authors Journal compilation ª 2007 FEBS 2847 Regulation of DRM sorting of Glut1 A T Sakyo et al A Intracellular loop FLAG chimera C13 (G1 : - 450) N C chimera N31 (G1 : 13 - 492) chimera N31C3 (G1 : 13-450) FLAG N 450 C N 13 C 450 13 492 Intracellular loop B B CHO Cell FG1 Transfection Fraction T S I FG3 N31C3 T S I T S I FLAG-M2 C CHO Cell Transfection FG1 Fraction T S I FG3 C13 N31 Glut3 T S I T S I T S I FLAG-M2 Glut3 Glut1 Glut1 β-Actin β-Actin Caveolin-1 α-Tubulin Fig Effect of replacing the N-terminal or C-terminal tail of Glut1 and Glut3 on DRM distribution (A) Schematic composition of FLAG–Glut1 ⁄ Glut3 chimeras Each construct contains the FLAG sequence DYKDDDDK inserted immediately after the methionine start codon In chimera C13, the last 42 amino acids of the C-terminal tail of Glut1 are replaced with the corresponding 48 amino acids of Glut3 By contrast, chimera N31 contains the first 12 amino acids of the N-terminal tail of Glut1 and, subsequently, amino acids 11–496 of Glut3 The location of the FLAG-tag is indicated by hatched areas at the N-terminus (B) CHO cells were transiently transfected with the C13 or N31 chimera, as described in Fig (B) After 48 h of transfection, the uptake of [3H]2DG was measured in the presence and absence of CB Absolute uptake values normalized to the quantity of protein expressed in CHO cells are given in nmoles per mg of protein per 10 One representative datum of several independent determinations is shown here DMSO, dimethylsulfoxide (C) Transiently transfected cells were solubilized by 0.5% Triton X-100 at °C and fractionation and immunoblotting for Glut1 and Glut3 were performed as described in Fig in the expression of these constructs, their distribution to the DRM fraction was distinctive As was seen with FG1 and endogenous Glut1, chimeric protein A was distributed to DRMs (Fig 5C) In contrast, 2848 Fig Effect of N-terminal and C-terminal substitution of Glut1 on DRM distribution (A) The FLAG-tagged chimera N31C3 contains the N-terminal and C-terminal amino acids of Glut3 and the backbone of Glut1 (B) CHO-K1 cells were transiently transfected with FG1, FG3 or N31C3, as described above Cells were solubilized by 0.5% Triton X-100 at °C, and fractionation and immunoblotting for Glut1, Glut3 and FLAG were performed as described in Fig chimeric protein B was distributed only to the soluble fraction, as seen with FG3 Increased glucose uptake was evident in the cells that overproduced chimera A in a CB-sensitive manner However, a small increase in glucose uptake was evident with the chimera B-transfected cells Role of a large intracellular loop of Glut1 in the DRM distribution The role of a large intracellular loop of Glut1 in the DRM distribution was further examined, as this domain was included in the most effective chimeric construct A (Fig 5), and is one of the major characteristics of these isoforms [25] However, chimeric construct D, in which this intracellular loop was replaced with Glut3, was not distributed to DRMs (Fig 6A) The shortening of this loop, i.e construct C, reduced the ability to distribute FEBS Journal 274 (2007) 2843–2853 ª 2007 The Authors Journal compilation ª 2007 FEBS T Sakyo et al Regulation of DRM sorting of Glut1 A A Intracellular loop Intracellular loop FLAG chimera C chimera A (G1 : 13-271) 13 chimera D chimera B 272 (G1 : 272-450) N (G1 : 13-206) 271 (G1 : 207-271) 450 B B C 206 13 N C 207 CHO Cell Transfection Fraction 271 FG1 FG3 chimera C chimera D T S I T S I T S I T S I FLAG-M2 Glut3 C CHO Cell Glut1 FG1 Transfection Fraction T S I FG3 chimera A chimera B T S I T S I T S I FLAG-M2 Glut3 β-Actin Fig Role of a large intracellular loop of Glut1 in DRM distribution (A) Chimera C contains amino acids 13–206 of Glut1 corresponding to the N-terminal half of the membrane-spanning segments and the backbone of Glut3 In chimera D, a large intracellular loop of Glut3 (amino acids 204–269) is replaced with the corresponding amino acids 207–271 of Glut1 (B) CHO-K1 cells were transiently transfected with these chimeras, and the distribution was determined after 48 h of transfection Glut1 Discussion β-Actin Fig Effects of the N-terminal half of the membrane-spanning segments and a large intracellular loop of Glut1A Chimera A contains amino acids 1–10 of Glut3, amino acids 13–271 of Glut1, and amino acids 291–496 of Glut3 Chimera B also contains amino acids 1–290 of Glut3, amino acids 272–450 of Glut1, and amino acids 449–496 of Glut3 The chimeras also have a FLAG epitope in the N-terminus (B) CHO cells were transiently transfected with the indicated FLAG-tagged chimeric construct, and the uptake of [3H]2DG was measured in the presence and absence of CB after 48 h of transfection Absolute uptake values normalized to the quantity of protein expressed in CHO cells are presented in nmoles per mg of protein per 10 The data are representative of three different experiments performed in duplicate DMSO, dimethylsulfoxide (C) The transfected CHO-K1 cells were solubilized by 0.5% Triton X-100 at °C, and fractionation and immunoblotting for Glut1 and Glut3 were performed as described in Fig DRMs, indicating some role for this loop region Further replacement within the N-terminal membrane-spanning regions of Glut1, included in chimera C, gave uncertain results The plasma membrane of mammalian cells is composed of functionally distinct membrane domains and their components This requires ordered gene expression as well as intricate post-translational sorting machinery that delivers proteins and lipids to the correct membrane domains during cell growth [29] The best characterized system comprises polarized epithelial cells, and the sorting machinery for membrane proteins that are recruited to different cell surfaces in polarized cells has been a subject of considerable interest Many studies have concentrated on identifying the determinants of basolateral and apical sorting signals at the molecular level [20,24,30] Heterogeneous membrane domains also exist in nonpolarized cells [8] These microdomains, called ‘lipid rafts’ or ‘DRMs’, because of their physicochemical nature, are enriched with ordered lipids such as cholesterol, glycolipids, and sphingolipids, which are present in cell membranes [31] Several proteins are preferentially distributed to these microdomains, including glycosylphosphatidylinositolanchored protein, the Src-family tyrosine kinases, FEBS Journal 274 (2007) 2843–2853 ª 2007 The Authors Journal compilation ª 2007 FEBS 2849 Regulation of DRM sorting of Glut1 T Sakyo et al heterotrimeric G proteins, and phospholipid-binding protein [21] Rafts constitute the scaffolding for signal transduction and several pathogens [22] We previously demonstrated the differential distribution of Glut isoforms Glut1 and Glut3 in the plasma membrane of nonpolarized HeLa cells and CHO-K1 cells Glut1 is distributed to raft-like DRMs, whereas Glut3 is predominantly found in fluid lipid domains [25] The distribution of Glut1 to DRMs in nonpolarized Clone cells [27] and 3T3-L1 cells [26] has also been reported However, the mechanism by which Glut1 but not Glut3 is recruited to DRMs is unknown It is well established that the C-terminus of Glut isoforms has various important roles in subcellular protein trafficking [24,32,33] and glucose uptake [34] In polarized epithelial cells, such as MDCK and Caco-2 cells, it has been shown that Gluts are, respectively, sorted to either the apical or basolateral surface Glut1 is principally found on the basolateral cell surface, whereas Glut3 is mainly recruited to the apical domain [18] Inukai et al have shown that Glut1 contains a basolateral sorting signal in its intracellular loop region [30], whereas the C-terminal tail of Glut3 contains a targeting motif directing the trafficking of basolateral-sorting Glut1 to the apical cell surface in MDCK cells [24] Recently, two proteins binding to the C-terminus of Glut1 have been reported One is the Glut1 transporter-binding protein Glut1CBP, which controls normal Glut1 trafficking in polarized epitherial cells, helping to regulate the level of Glut1 in the plasma membrane [32,33] The other is stomatin, a type membrane protein that interacts with the C-terminus of Glut1 in DRMs of Clone cells [26,35] We therefore speculated that the C-terminus of Glut1 has some role in the DRM distribution However, in the present study, we observed that replacement of neither the C-terminal nor the N-terminal amino acids of Glut1 domains with the corresponding amino acids of Glut3 had any significant effect on the distribution of Glut1 in CHO-K1 cells (Fig 3A) The expression levels of these chimeric proteins and enhanced glucose uptake were not greatly affected as compared to those in the vector-transfected control cells The results suggested that a Glut1 ⁄ Glut3 chimera that has the C-terminus of Glut3 can be trafficked to the cell surface and distributed to DRMs like Glut1 Analysis of chimeric constructs A and B demonstrated that a large region from the N-terminal half of TM1 to the large cytoplasmic loop of Glut1 is necessary for the DRM distribution (Fig 5C) Further shortening of this region or replacement of the large cytoplasmic loop of Glut1 with the appropriate region of Glut3 clearly affected the distribution to DRMs (Fig 6B), and a 2850 chimeric analysis within these regions provided indefinite results Thus, our data suggest that the regulatory elements for the DRM distribution of Glut1 in nonpolarized cells are different from those for the apical ⁄ basolateral sorting signals of Glut1 and Glut3 in polarized epitherial cells Rather, the DRM distribution may require a specific tertiary structure to be oriented in liquid-ordered lipid phases Some recent reports have discussed the biological significance of the DRM distribution of Glut1, suggesting that the redistribution of Glut1 among different microdomains of the plasma membrane in nonpolarized cells may have a role in the stressinduced activation of glucose transport [27,32,36] The results imply that the distribution to the DRM of Glut1 in nonpolarized cells is closely related to several regulatory systems for glucose transport, which might be distinct from those in polarized cells Further studies are needed to clarify the molecular mechanism by which Glut1 is distributed to DRM domains, and its physiologic roles under various conditions Experimental procedures Antibodies and reagents The rabbit polyclonal antibody against C-terminal peptides of human Glut1 was purchased from Millipore (Billerica, MA, USA) The rabbit polyclonal antibody to Glut3 was purchased from Medical & Biological Laboratories (Nagoya, Japan) The mouse monoclonal antibodies to b-actin, a-tubulin and FLAG M2 were from Sigma (St Louis, MO, USA), and the mouse monoclonal antibodies to human caveolin-1 were from BD Biosciences (Bedford, MA, USA) An enhanced chemiluminescence (ECL) kit and [3H]2-deoxy-d-glucose ([3H]2DG) (1 lCi mL)1) were obtained from GE Healthcare (Chalfont St Giles, UK) CB was provided by Sigma and Calbiochem (La Jolla, CA, USA) Plasmids The wild-type Glut1–enhanced green fluorescent protein (EGFP) and wild-type Glut3–EGFP cDNA constructs were prepared by subcloning the full-length rabbit Glut1 or human Glut3 cDNA into the Bgl2-Xho1 site of the vector pEGFP-C2 (Clontech, Mountain View, CA, USA) to generate N-terminal EGFP fusion proteins FG1, FG3 and FLAG-tagged Glut1 ⁄ Glut3 chimeric cDNAs were produced according to previously described methods [39] A pUC ⁄ Glut1 or pSRa ⁄ Glut3 vector [11] was used as a template for PCR The mammalian expression vector pCMV-Script was kindly provided by O Kuge (School of FEBS Journal 274 (2007) 2843–2853 ª 2007 The Authors Journal compilation ª 2007 FEBS T Sakyo et al Sciences, Kyushu University) cDNAs encoding FG1, FG3 and chimeric Gluts were ligated into the Sal1–Not1 site of pCMV All of the FLAG-tagged constructs have the sequence MDYKDDDDK inserted after the first methionine Inserts were fully sequenced, and were observed to have no unexpected mutations The six chimeras had the compositions shown in Fig Cell culture and transfection CHO-K1 cells were cultured in F-12 medium (Invitrogen, Carlsbad, CA, USA) containing 10% fetal bovine serum (MBL, Nagoya, Japan), penicillin (100 mL)1) and streptomycin (100 lgỈmL)1) under humidified 5% CO2 ⁄ 95% air at 37 °C, as described previously [40] These cells were free from mycoplasma contamination Lipofectamine reagent and Opti-MEM were purchased from Invitrogen One day before transfection, CHO-K1 cells were trypsinized and seeded onto 100 mm plastic culture dishes at · 106 cells per dish On the following day, transfection procedures were performed using 30 lL of lipofectamine diluted in 70 lL of Opti-MEM (Gibco-BRL) and lg of Glut ⁄ pCMV plus lg of GFP diluted in 100 lL of supplemental Opti-MEM in 100 mm dishes Cells were incubated in the presence of the lipofectamine ⁄ DNA mixture for h at 37 °C, in 5% CO2, and then incubated overnight in F-12 medium in the presence of 10% fetal bovine serum At 48 h post-transfection, cells were used for immunoblotting and immunofluorescence analysis as described For 2DG uptake assays, the cells were seeded in cm dishes at · 105 cells per dish Immunofluorescence analysis CHO-K1 cells were transfected with GFP-tagged Glut1 and Glut3, as described under ‘Cell culture and transfection’ At 24 h post-transfection, cells were seeded in a glass-based dish and incubated overnight at 37 °C Living cells were visualized by confocal microscopy (LSM 510, Carl Zeiss Microimaging, Jena, Germany) Digital images were processed with photoshop (Adobe, San Jose, CA, USA) Detergent solubilization and immunoblotting The cells growing in two 10 cm dishes (CHO-K1 cells, about · 107 cells per dish) were washed once with cold NaCl ⁄ Pi and scraped They were then centrifuged for 10 at 180 g using a swinging bucket rotor RS-240, 2100 (Kubota, Tokyo, Japan), and washed with Hepes buffer After clarification by centrifugation at 280 g using a TMA-4 rotor, MRX-150 (TOMY, Tokyo, Japan) for min, pellets were treated with 0.1–0.5 mL of Hepes buffer containing 0.5% Triton X-100 (Sigma), 10 mm sodium Hepes, 150 mm NaCl, mm EDTA and 0.5 mm Regulation of DRM sorting of Glut1 phenylmethanesulfonyl fluoride for 30 at °C, as described previously [25] An aliquot of the treated cells was preserved as the total fraction (T) The remainder was centrifuged at 13 000 g for 30 at °C using a TMA-4 rotor, MRX-150, and the supernatant was used as the soluble fraction (S) The pellet was washed in mL of cold Hepes buffer without Triton X-100, and was centrifuged at 13 800 g for 10 at °C using a TMA-4 rotor, MRX-150 The pellet (insoluble fraction, I) was solubilized with 0.1–0.5 mL of lysis buffer, containing 10 mm Tris ⁄ HCl, 150 mm NaCl, 1% Triton X-100, 0.5% Nonidet P-40, mm EDTA, mm EGTA, and 0.5 mm phenylmethanesulfonyl fluoride (pH 7.5) The protein concentration was determined using bicinchoninic reagent (Pierce, Rockford, IL, USA) with BSA as a standard Protein samples (10 lg) were subjected to 10% SDS ⁄ PAGE, and transferred to Immobilon-P membranes (Millipore), which were incubated in NaCl ⁄ Tris ⁄ Tween (500 mm NaCl, 20 mm Tris ⁄ HCl, pH 7.5, plus 0.1% Tween-20) containing 5% skimmed milk (Sanko Junyaku, Tokyo, Japan), followed by rabbit polyclonal antibody or mouse monoclonal antibody (1 : 1000–2000) The membranes were further incubated with horseradish peroxidase-conjugated anti-(rabbit IgG) or anti-(mouse IgG) serum (Amersham Pharmacia Biotech), and visualized with the ECL detection kit [3H]2DG uptake assays CHO cells were transiently transfected with lg of chimeric forms of Glut1 and Glut3, as described in ‘Cell culture and transfection’ Duplicate culture plates were washed with NaCl ⁄ Pi, and then incubated for 10 in glucose uptake medium consisting of mL of glucose-free DMEM (Sigma) containing 2.5 lCi of [3H]2DG, mm 2DG and 10 lL of either dimethylsulfoxide alone or dimethylsulfoxide containing CB at a final concentration of 10 lm, as previously described [11,41] Uptake was terminated by removal of the medium followed by three rapid washes with mL of NaCl ⁄ Pi Then, cells were incubated with 5% trichloroacetic acid for more than 20 at °C, and cellular radioactivity was determined by liquid scintillation counting For protein extraction, cells were washed with mL of NaCl ⁄ Pi and incubated with 0.5 m NaOH for 30 at 37 °C The protein concentration was determined using bicinchoninic acid reagent with BSA as a standard Acknowledgements We are grateful to Dr K Ishidate for encouragement and advice We thank Dr O Kuge for helpful suggestions regarding the construction of FLAG-tagged chimeric constructs We also thank Toshie Gamou for technical assistance in chimeric construction, and FEBS Journal 274 (2007) 2843–2853 ª 2007 The Authors Journal compilation ª 2007 FEBS 2851 Regulation of DRM sorting of Glut1 T Sakyo et al Yumi Ikeda for assistance in the transfection and immunoblotting This study was supported in part by the Human Science Foundation of Japan (T Kitagawa) and the Japan Science Society, Sasagawa Foundation (T Sakyo) 13 References Bell GI, Burant CF, Takeda J & Gould GW (1993) Structure and function of mammalian facilitative sugar transporters J Biol Chem 268, 19161–19164 Joost HG & Thorens B (2001) The extended GLUTfamily of sugar ⁄ polyol transport facilitators: 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development Biochem Biophys Res Commun 154, 1204–1211 FEBS Journal 274 (2007) 2843–2853 ª 2007 The Authors Journal compilation ª 2007 FEBS 2853 ... replacement of neither the C-terminal nor the N-terminal amino acids of Glut1 domains with the corresponding amino acids of Glut3 had any significant effect on the distribution of Glut1 in CHO-K1 cells. .. basolateral-sorting Glut1 to the apical cell surface in MDCK cells [24] Recently, two proteins binding to the C-terminus of Glut1 have been reported One is the Glut1 transporter- binding protein Glut1CBP,... normal Glut1 trafficking in polarized epitherial cells, helping to regulate the level of Glut1 in the plasma membrane [32,33] The other is stomatin, a type membrane protein that interacts with the